DESCRIPTION: The research program addresses questions of fundamental importance to human health-biological design rules that determine whether cells respond to chemotactic signals by disrupting intercellular contacts in ways that fundamentally impact the organization and integrity of tissues. This research exploits the enabling tools of soft lithography and live cell imaging to define quantitative biological design rules that control cellular decisions. Specifically, we will (i) define quantitatively how concentration profiles of an immobilized, intercellular adhesive cue, cadherin govern the differential migratory response (haptotaxis) of normal and malignant mammary epithelial cells, (ii) identify the impact of chemotactic gradients on epithelial cell polarity and migration, and (iii) determine how cadherin ligation and chemoattractants coordinately regulate the spatiotemporal distributions of GTPase activities that direct cell migration. Soft lithography enables the establishment of defined fields of adhesive and chemotactic cues in ways that are not accessible in vivo or in standard tissue culture format. To this end, we will uniquely quantify cell migratory responses to patterns of adhesive and soluble cues-individually or in combination-and define synergistic or antagonistic interactions regulating cell outcomes. Live cell imaging will then directly determine how spatially and temporally distributed extracellular cues alter zones of signaling activities within single cells and thus bridge the gap between external stimuli, global cell response, and fundamental intracellular biochemical changes. If successful, the proposed strategies in this R21 proposal will uniquely identify mechanisms governing cell adhesion and chemotaxis in cancer and in development. The validation of these approaches will lay the foundation for future investigations of additional factors such as integrins, growth factors, substrate stiffness, or any number of parameters relevant to human development and disease. This multidisciplinary team possesses core competencies in microfabrication (Nuzzo), surface modification and protein immobilization (Leckband), biochemical/cell biological techniques (Wang), as well as expertise in cadherin biology (Leckband) and chemotaxis (Wang) essential for the success of this program. Nuzzo and Leckband have worked together on multiple projects over several years. Leckband and Wang are collaborating on an independent project involving cadherins and stem cell differentiation. Wang frequently advises Leckband group members on experimental protocols for cell work, and Wang and Leckband periodically hold joint group meetings. The labs and offices of all three PIs are in adjacent buildings. This proposal results from several conversations between the PIs. Public Health Relevance: Cell migration is an essential morphogenetic process in tissue formation, repair, and regeneration. It also drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. In tissues, cell movements in both normal and diseased tissues involve the coordinated regulation of cell motility and cell-cell adhesion. Cell motility machinery is often triggered by soluble growth factors and chemoattractants, with a concomitant destabilization of intercellular adhesion. Furthermore, gradients of both chemoattractants and adhesion are thought to guide cell movements in tissues. It is the interplay between signals governing chemotaxis or cell migration versus cell-cell adhesion that ultimately governs the formation and structural integrity of tissues. Understanding these processes in both normal development and disease progression is predicated on elucidating the biological design rules that regulate the balance between firm intercellular adhesion and migration. Despite the importance of this issue to human health, these questions have received very little attention. We postulate that the current knowledge gap is due to the limitations inherent in current approaches used to study cell adhesion, migration, and chemotaxis as well as the lack of approaches capable of quantitatively evaluating the impact of adhesive and diffusive signals on cell behavior. This multidisciplinary team embodies the core capabilities needed to address this complex problem. Specifically, current competencies in soft lithography, cell adhesion, and cell migration enable the generation of quantitatively defined biochemical cues-both in immobilized and diffusive form-in order to quantitatively define the balance of different signals that mutually regulate cell behavior. Importantly, these devices enable the quantitative definition of biochemical signals in ways that are not accessible in vivo or in standard tissue culture format. This level of control is essential for defining, for example, chemoattractant in uniform or gradient forms that destabilize cell-cell junctions to promote migration. To this end, this proposed research will use these devices to uniquely quantify cell migratory responses to patterns of adhesive and soluble cues-individually or in combination-in order to define synergistic or antagonistic interactions between chemotactic and adhesive biochemical cues that regulate cell outcomes. We will combine these patterning tools with live cell imaging to directly bridge the gap between external stimuli and the fundamental intracellular biochemical processes that underlie global cell response. This unique complementation of engineered cellular environments and real-time spatiotemporal imaging of biochemical processes should establish causal relationships between spatially and temporally distributed extracellular cues, the intracellular coordination of zones of signaling activities and protein distributions within single cells, and global cell response. Importantly, such real-time spatiotemporal information is also not accessible with standard biochemical assays. The knowledge generated by these studies will be directly relevant to human health and may identify effective therapies for the treatment and prevention of cancer, for example. Furthermore, if successful, the general validation of these approaches will lay the foundation for further studies of additional biochemical factors or environmental parameters-as single components or in various combinations-including integrins, growth factors, substrate stiffness, or any number of parameters relevant to human development and disease.